T cells recognize peptides bound to MHC molecules using a clonotypic receptor (TCR). Depending on the differentiation state of the T lymphocyte (i.e., responsiveness to a second signal) and/or the context in which the antigenic peptide is presented (i.e., availability of a second signal), this interaction can initiate distinct molecular programs leading to opposite outcomes (T cell activation, unresponsiveness, or death). A precise regulation of these responses results in a system that is both immunocompetent (able to be activated by and eliminate dangerous microorganisms) and self tolerant (able to eliminate or anergize T cell populations bearing TCRs specific for self antigens). The main process through which immunotolerance is achieved involves elimination of potentially dangerous T cells (negative selection or clonal deletion). A number of studies have shown that negative selection is mediated by programmed cell death (apoptosis) and can be blocked by inhibitors of macromolecular synthesis, indicating that mRNA(s) with a short half-life encoding a protein(s) with high turnover rate and/or that synthesis of new mRNA(s) and its translation into protein(s) is required. Our major objective is the dissection of the intracellular events linking the TCR/Ag+MHC interaction to cell death. Specifically, the project will progress in two distinct but connected stages: 1) isolation and identification of the genes involved in negative selection and 2) characterization of the biochemical processes responsible for their functional activation. Our strategic approach is based on the hypothesis that programmed cell death could be blocked by specifically inhibiting translation of required mRNAs. This goal can be achieved by expressing in the cells adequate levels of specific antisense RNAs. Since this hypothesis cannot be tested in vivo, we have used a mouse T cell hybridoma (3DO) which undergoes programmed cell death upon TCR triggering. 3DO's apoptosis, as in negative selection of normal T cells, requires ongoing mRNA transcription and protein synthesis. Given the similarities between TCR-induced 3DO cell death and negative selection, this hybridoma can be used as an in vitro model for the initial identification and characterization of genes involved in the deletion of self-reactive T cells. To this end, we have constructed cDNA libraries into eukaryotic expression vectors using an mRNA source enriched in apoptotic genes. Upon transfection of the libraries, 3DO has been triggered for apoptosis. If our assumpion is correct, cells transfected with plasmids encoding cDNAs necessary to carry out the death process and expressing antisense RNAs should be rescued from TCR-induced death. Therefore, the plasmids recovered from surviving cells should contain "apoptotic genes." Once defined, the requirements for the functional activation of these molecules will be studied in more detail. The physiologic relevance of any finding must be subsequently confirmed in an in vivo animal model.